The bicyclic .gamma.-lactam, 2-azabicyclo[2.2.1]hept-5-en-3-one, is a useful synthon that can be used for the production of carbocyclic nucleosides which are gaining in importance as therapeutic agents. Published areas to which such nucleosides are being targeted include antivirals (e.g. Vince and Hua, J. Med. Chem., 33:17-21 (1990), against e.g. HIV) and cardiac vasodilators (adenosine agonists). A major benefit of the carbocyclic ring in such agents is its resistance to breakdown by enzymes in the body. By comparison, naturally-occurring ribosyl nucleosides may be more readily cleaved by nucleases, so that their bioactivity is lost.
Although carbocyclic nucleosides are known in nature, e.g. Aristeromycin from Streptomyces citricolor, natural yields tend to be low and the isolated products have then to be further manipulated to obtain more useful compounds. A more economic route is to synthesise the required compounds chemically, starting from the .gamma.-lactam. However, as chemically synthesised, .gamma.-lactam is racemic. By conventional synthesis, the ultimate drug will also be a mixture of enantiomers, which causes regulatory concerns if one of the enantiomers is not very active or causes unwanted side-effects. There is a need therefore to put a step into the synthesis where either of the two enantiomers of a racemic synthon can be isolated and the rest of the drug then built on it.
An effective way of doing this is to use an enzyme to selectively hydrolyse one enantiomer of the racemic .gamma.-lactam across the amide bond, to give the cyclic amino acid compound and leave the other enantiomer. The remaining lactam can then be readily separated from the amino acid product by extraction into dichloromethane, purified by crystallisation and used in subsequent downstream chemistry to build up the required drug. By careful selection of the right enzyme it is possible to find an enzyme highly selectively for only one of the lactam enantiomers such that at marginally greater than 50% conversion, lactam of high ee (&gt;90%) remains. Enzymes have been found that are selective for either of the two enantiomers.
EP-A-0424064 discloses methods for carrying out the above described resolution and provides two organisms that produce enzymes that have the different selectivities. A Rhodococcus strain produces an enzyme which hydrolyses the (-) lactam, enabling the (+) lactam to be isolated for further use, whereas a Pseudomonad produces an enzyme which hydrolyses the (+) lactam, enabling isolation of the (-) lactam.
Further enzymes that carry out these active hydrolyses have also been described in the literature. Thus Taylor et al, Tetrahedron; Asymmetry, 4(6):1117-1128 (1993), describe an enzyme selective for hydrolysis of the (+) lactam from Pseudomonas fluorescens and an enzyme selective for the (-) lactam from a strain of Aureobacterium. A further enzyme selective for the hydrolysis of the (+) lactam has been described by Brabban et al. J. Ind. Microbiology. 16:8-14 (1996).
In order to develop a robust industrial biotransformation process, it is desirable to use an enzyme or whole cell biocatalyst that is relatively stable. This can enable biocatalyst recycling and re-use through immobilisation, thus greatly reducing biocatalyst cost and enabling handling of the biocatalyst on a large scale without significant losses of activity. It is also often found that more stable biocatalysts are better able to tolerate high substrate and/or product concentrations without inactivation. This then enables biotransformations to be run at the highest concentration of reactants possible, given kinetic and handling constraints. This has two advantages: it results in minimal reactor volume requirements and also minimises liquid handing volumes during product work-up.
Taylor et al, supra, describe a lactamase from Aureobacterium species that is very stable at elevated temperatures and which selectively hydrolyses the (-) .gamma.-lactam, giving the (+) .gamma.-lactam and (-) amino acid as a product. The enzyme from this organism has been immobilised and maintains its stability over months of operation. No enzyme with good stability and the opposite selectivity is known, although Brabban et al, supra, screened a number of different potential isolates. Previous work with Pseudomonad type organisms displaying the required lactamase activity had shown them to have poor stability. This is unfortunate since it is the (-) .gamma.-lactam which is the more useful synthon, having the more natural stereochemistry and making it easier to build up functionality than for instance the (-) amino acid formed by the action of the Aureobacterium lactamase. There is therefore a need for a stable .gamma.-lactamase with high selectivity for the hydrolysis of the (+) bicyclic .gamma.-lactam.